Glycated human DNA is a preferred antigen for anti-DNA antibodies in diabetic patients

Aims: Glycation of proteins and DNA, results in the generation of free radicals causing structural modification of biomacromolecule. This leads to the generation of neo-antigenic epitopes having implication in diabetes mellitus. In this study, human placental DNA was glycated with fructose and its binding was probed with the serum antibodies from type 1 and 2 diabetes patients. Methods: Glycation was carried out by incubating DNA (10 mu g/ml) with fructose (25 mM) for 5 days at 37 degrees C. The induced structural changes in DNA were studied by spectroscopic techniques, thermal denaturation studies and agarose gel electrophoresis. Furthermore, binding characteristics of autoantibodies in diabetes (type 1 and 2) patients were assessed by direct binding and competitive ELISA. Results: DNA glycation with fructose resulted in single strand breaks, hyperchromicity in UV spectrum and increased fluorescence intensity. Thermal denaturation studies demonstrated the unstacking of bases and early onset of duplex unwinding. Type 1 diabetes patients exhibited enhanced binding with glycated DNA as compared to native form, while for type 2 diabetes only those with secondary complications (Nephropathy) showed higher binding. Conclusions: Glycation of DNA has resulted in structural perturbation causing generation of neo-antigenic epitopes that are better antigens for antibodies in diabetes patients. (C) 2011 Elsevier Ireland Ltd. All rights reserved.